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D R Dean

Publications and source records attributed to D R Dean.

64 records · Page 4Linked to original sources
PubMed1988

Isolation, sequencing, and mutagenesis of the nifF gene encoding flavodoxin from Azotobacter vinelandii.

The nifF gene encoding flavodoxin from Azotobacter vinelandii OP was cloned and its DNA sequence determined. It is located adjacent to, or possibly within, the major nif cluster and it is preceded by nif-specific regulatory elements. Southern hybridization analysis revealed that there is only a single copy of the nifF gene on the A. vinelandii OP genome. Mutant strains were constructed which have an insertion mutation or an insertion and a deletion mutation within the nifF gene coding sequence. These mutant strains are capable of diazotrophic growth, indicating that flavodoxin is not the unique physiological electron donor to nitrogenase. The results of nifF-lacZYA gene fusion experiments and Northern hybridization analyses indicated that the nifF gene is both transcribed and translated under nitrogen fixing and non-nitrogen fixing conditions. However, under nitrogen fixing conditions a substantial increase in both nifF synthesis and in accumulation of an approximately 800-base pair nifF-encoding mRNA species was observed. Furthermore, strains mutated within the nifF gene have only 70% of the wild type in vivo nitrogenase activity as determined by whole cell acetylene reduction assays. These data demonstrate that the nifF-encoded flavodoxin of A. vinelandii OP, although not essential for nitrogen fixation, is required for maximum in vivo nitrogenase activity.

Amino Acid Sequence↗
PubMed1988

Nucleotide sequence and mutagenesis of the nifA gene from Azotobacter vinelandii.

The nucleotide sequence of the nifA gene from Azotobacter vinelandii was determined. This gene encodes an Mr = 58,100 polypeptide that shares significant sequence identity when compared to nifA-encoded products from other organisms. Interspecies comparisons of nifA-encoded products reveal that they all have a consensus ATP binding site and a consensus DNA binding site in highly conserved regions of the respective polypeptides. The nifA gene immediately precedes the nifB-nifQ gene region but is unlinked to the major nif gene cluster from A. vinelandii. A potential regulatory gene precedes and is apparently cotranscribed with nifA. Mutant strains that have a deletion or a deletion plus an insertion within nifA are incapable of diazotrophic growth and they fail to accumulate nitrogenase structural gene products.

Amino Acid Sequence↗
PubMed1987

Iron-molybdenum cofactor biosynthesis in Azotobacter vinelandii requires the iron protein of nitrogenase.

Nitrogenase is composed of two separately purified proteins called the Fe protein and the MoFe protein. In Azotobacter vinelandii the genes encoding these structural components are clustered and ordered: nifH (Fe protein)-nifD (MoFe protein alpha subunit)-nifK (MoFe protein beta subunit). The MoFe protein contains an ironmolybdenum cofactor (FeMo cofactor) whose biosynthesis involves the participation of at least five gene products, nifQ, nifB, nifN, nifE, and nifV. In this study an A. vinelandii mutant strain, which contains a defined deletion within the nifH (Fe protein) gene, was isolated and studied. This mutant is still able to accumulate significant amounts of MoFe protein subunits. However, extracts of this nifH deletion strain have only very low levels of MoFe protein acetylene reduction activity. Fully active MoFe protein can be reconstituted by simply adding isolated FeMo cofactor to the extracts. Fe protein is not necessary to stabilize or insert this preformed FeMo cofactor into the FeMo cofactor-deficient MoFe protein synthesized by the nifH deletion strain. Extracts of the nifH deletion strain can carry out molybdate and ATP-dependent in vitro FeMo cofactor biosynthesis provided Fe protein is added, demonstrating that they contain the products encoded by the FeMo cofactor biosynthetic genes. These data demonstrate that the Fe protein is physically required for the biosynthesis of FeMo cofactor in A. vinelandii.

Azotobacter↗
PubMed1987

Site-directed mutagenesis of the nitrogenase MoFe protein of Azotobacter vinelandii.

A strategy has been formulated for the site-directed mutagenesis of the Azotobacter vinelandii nifDK genes. These genes encode the alpha and beta subunits of the MoFe protein of nitrogenase, respectively. Six mutant strains, which produce MoFe proteins altered in their alpha subunit by known single amino acid substitutions, have been produced. Three of these transversion mutations involve cysteine-to-serine changes (at residues 154, 183, and 275), two involve glutamine-to-glutamic acid changes (at residues 151 and 191), and one involves an aspartic acid-to-glutamic acid change (at residue 161). All three possible phenotypic responses are observed within this group- i.e., normal, slow, and no growth in the absence of a fixed-nitrogen source. Two-dimensional gel electrophoresis indicates that all mutants accumulate normal levels of the subunits of both nitrogenase component proteins. Whole-cell and crude-extract acetylene-reduction activities indicate substantial levels of Fe protein activity in all strains. In contrast, MoFe protein activities do not parallel the diazotrophic growth capability for all strains. Two strains appear to exhibit altered substrate discrimination. Such analyses should aid in the identification of metallocluster-binding sites and subunit-subunit interaction domains of the MoFe protein and also provide insight into the mechanistic roles of the various prosthetic groups in catalysis.

Journal Article↗
PubMed1987

Products of the iron-molybdenum cofactor-specific biosynthetic genes, nifE and nifN, are structurally homologous to the products of the nitrogenase molybdenum-iron protein genes, nifD and nifK.

The genes from Azotobacter vinelandii, which are homologous to the iron-molybdenum cofactor biosynthetic genes, nifE and nifN, from Klebsiella pneumoniae, have been cloned and sequenced. These genes comprise a single transcription unit and are located immediately downstream from the nitrogenase structural gene cluster (nifHDK). DNA sequence analysis has revealed that the products of the nifE and nifN genes contain considerable homology when compared with the nifD (MoFe protein alpha subunit) and the nifK (MoFe protein beta subunit) gene products, respectively. These striking sequence homologies indicate a structural and functional relationship between a proposed nifEN product complex and the nitrogenase MoFe protein as well as imply an ancestral relationship between these gene clusters. The isolation and characterization of strains which contain deletions within the nifEN gene cluster demonstrate a role for these products in iron-molybdenum cofactor biosynthesis in A. vinelandii.

Amino Acid Sequence↗
PubMed1986

Activity, reconstitution, and accumulation of nitrogenase components in Azotobacter vinelandii mutant strains containing defined deletions within the nitrogenase structural gene cluster.

The Azotobacter vinelandii genes encoding the nitrogenase structural components are clustered and ordered: nifH (Fe protein)-nifD (MoFe protein alpha subunit)-nifK (MoFe protein beta subunit). In this study various A. vinelandii mutant strains which contain defined deletions within the nitrogenase structural genes were isolated and studied. Mutants deleted for the nifD or nifK genes were still able to accumulate significant amounts of the unaltered MoFe protein subunit as well as active Fe protein. Extracts of such nifD or nifK deletion strains had no MoFe protein activity. However, active MoFe protein could be reconstituted by mixing extracts of the mutant strains. These results establish an approach for the purification of the individual MoFe protein subunits. Mutants lacking either or both of the MoFe protein subunits were still able to synthesize the iron-molybdenum cofactor (FeMo-cofactor), indicating that in A. vinelandii the FeMo-cofactor is preassembled and inserted into the MoFe protein. In contrast, a mutant strain lacking both the Fe protein and the MoFe protein failed to accumulate any detectable FeMo-cofactor. The further utility of specifically altered A. vinelandii strains for the study of the assembly, structure, and reactivity of nitrogenase is discussed.

Azotobacter↗
PubMed1985

Complete nucleotide sequence of the Azotobacter vinelandii nitrogenase structural gene cluster.

DNA fragments coding for the structural genes for Azotobacter vinelandii nitrogenase have been isolated and sequenced. These genes, nifH, nifD and nifK, code for the iron (Fe) protein and the alpha and beta subunits of the molybdenum-iron (MoFe) protein, respectively. They are arranged in the order: promoter:nifH:nifD:nifK. There are 129 nucleotides separating nifH and nifD and 101 nucleotides separating nifD and nifK. The amino acid (aa) sequences deduced from the nucleotide sequences are discussed in relation to the prosthetic group-binding regions of the nifHDK-encoded polypeptides.

Amino Acid Sequence↗
PubMed1985

Azotobacter vinelandii nifD- and nifE-encoded polypeptides share structural homology.

The Azotobacter vinelandii nifE gene was isolated and its complete nucleotide sequence was determined. The amino acid sequences deduced from the A. vinelandii nifE and nifD gene sequences were compared and found to share striking primary sequence homology. This homology implies a functional and possibly an evolutionary relationship between these two gene products. The structural homology is discussed with regard to the potential FeMo cofactor binding properties of these polypeptides and the possible role of a nifEN product complex as a surrogate MoFe protein.

Journal Article↗
PubMed1977

Alteration of the Bacillus subtilis glutamine synthetase results in overproduction of the enzyme.

A mutational leading to glutamine auxotrophy was located near a 5-fluorouracil resistance marker in the citB-thyA region of the Bacillus subtilis chromosome. This mutation resulted in a glutamine synthetase with altered kinetic and feedback properties. The specific activity of manganese-stimulated glutamine synthetase activity in crude extracts was 18-fold higher, and the magnesium-stimulated activity was about 30% that of the wild type. Quantitation of the enzyme by precipitation with antibody prepared against pure enzyme confirmed the presence of high enzyme levels in the mutant. This mutation is very closely linked (recombination index of 0.03) to another glutamine auxotroph containing enzyme with altered electrophoretic and heat sensitivity properties. Mutations in the structural gene for glutamine synthetase may result not only in altered catalytic and regulatory properties but also in altered production of the enzyme.

Alanine↗